内质网应激抑制剂4-PBA对高糖诱导大鼠视网膜Müller细胞胶质增生的作用及机制  

Effect of endoplasmic reticulum sress inhibitor 4-PBA on high glucose-induced proliferation of rat Müller cells and its mechanism

作　　者：酆啸 龙秋双 甘诗泉 沈祥春 陈妍 FENG Xiao;LONG Qiushuang;GAN Shiquan;SHEN Xiangchun;CHEN Yan(School of Pharmaceutical Sciences,Guizhou Medical University&The High Educational Key Laboratory of Guizhou Province for Natural Medicinal Pharmacology and Druggability,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州医科大学药学院&贵州省高等学校天然药物药理与成药性评价重点实验室,贵州贵阳550025

出　　处：《贵州医科大学学报》2022年第12期1365-1371,1402,共8页Journal of Guizhou Medical University

基　　金：国家自然科学基金(82060772);贵州省科学技术基金重点项目(黔科合基础[2020]1Z069);贵州省科技计划项目基础研究重点项目(黔科合基础[2019]1438);贵州医科大学国家自然科学基金培育项目(19NSP072)。

摘　　要：目的探讨内质网应激(ERS)抑制剂4-苯基丁酸(4-PBA)对高糖(HG)诱导大鼠视网膜Müller细胞胶质增生的作用及机制。方法取对数生长期Müller细胞,选择0、0.1、0.25、0.5、1、2.5、5、7.5、10 mmol/L 4-PBA预处理细胞1 h,DMEM培养基继续培养24 h和48 h后,采用二苯基四氮唑溴盐(MTT)法检测Müller细胞存活率;对数生长期Müller细胞分为Control组(5.5 mmol/L葡萄糖)、Mannitol组(34.5 mmol/L甘露醇+5.5 mmol/L葡萄糖)、HG组(40 mmol/L葡萄糖)、4-PBA-L组(1 mmol/L 4-PBA+40 mmol/L葡萄糖)及4-PBA-H组(5 mmol/L 4-PBA+40 mmol/L葡萄糖),各组细胞分别给与相应处理,4-PBA-L和4-PBA-H组给予不同浓度4-PBA预作用1 h,再给予40 mmol/L葡萄糖作用细胞48 h;处理结束后,采用Giemsa染色法观察各组Müller细胞的形态学特征,采用流式细胞术检测各组Müller细胞活性和细胞周期;采用蛋白质免疫印记法(Western blot)检测各组Müller细胞中葡萄糖调节蛋白78(GRP78)、蛋白激酶R样内质网激酶(PERK)、磷酸化-PERK(p-PERK)、真核翻译起始因子2α(eIF2α)、磷酸化-eIF2α(p-eIF2α)、转录激活因子(ATF4)、胶质纤维酸性蛋白(GFAP)及血管内皮生长因子(VEGF-A)的蛋白表达。结果HG组Müller细胞较Control组数目急剧增多、细胞形状变圆、细胞间隙变小,4-PBA-L、4-PBA-H组Müller细胞数较HG组减少、细胞形状恢复长条形、间隙变宽;MTT检测结果显示,HG组Müller细胞的存活率和细胞周期S期水平较Control组增加(P<0.05),4-PBA-L、4-PBA-H组Müller细胞存活率和细胞周期S期水平则较HG组降低(P<0.05);Western blot检测结果显示,HG组Müller细胞中GRP78、p-PERK、p-eIF2α、ATF4、GFAP及VEGF-A蛋白表达较Control组升高(P<0.05),4-PBA-L、4-PBA-H组Müller细胞中GRP78、p-PERK、p-eIF2α、ATF4、GFAP及VEGF-A蛋白表达则较HG组降低(P<0.05)。结论4-PBA可抑制HG诱导大鼠视网膜Müller细胞的异常增殖并降低细胞的胶质增生水平,其作用机制可能与调节ERS�Objective To investigate the effect of endoplasmic reticulum stress(ERS)inhibitor 4-phenylbutyric acid(4-PBA)on high glucose(HG)-induced proliferation of rat retinal Müller cells and its mechanism.Methods Müller cells at logarithmic growth phase were treated with 0,0.1,0.25,0.5,1,2.5,5,7.5,and 10 mmol/L 4-PBA for 1 h,respectively,cultured with the DMEM medium for 24 h and 48 h,respectively.Methylthiazolyldiphenyl-tetrazolium bromide(MTT)assay was used to determine Müller cell survival rate.Müller cells at logarithmic growth phase were divided into control group(5.5 mmol/L glucose),Mannitol group(34.5 mmol/L mannitol+5.5 mmol/L glucose),HG group(40 mmol/L glucose),4-PBA-L group(1 mmol/L 4-PBA+40 mmol/L glucose),and 4-PBA-H group(5 mmol/L 4-PBA+40 mmol/L glucose)based on treatment reagents and dosage.4-PBA-L and 4-PBA-H groups were pretreated with 4-PBA at different concentrations for 1 h,then 40 mmol/L glucose for 48 h.After treatment,Giemsa staining was used to observe the morphological characteristics of Müller cells in each group,and flow cytometry was used to detect the activity and cell cycle of Müller cells in each group.Western blot was used to detect the protein expression levels of glucose regulated protein 78(GRP78),protein kinase R-like endoplasmic reticulum kinase(PERK),phosphorylated PERK(p-PERK),and eukaryotic translation initiation factor 2α(eIF2α),phosphorylated-eIF2α(P-eIF2α),activating transcription factor 4(ATF4),glial fibrillary acidic protein(GFAP),and vascular endothelial growth factor(VEGF-A)in Müller cells in each group.Results When compared with control group,the number of Müller cells in HG group was sharply increased,and cell shape became round,and the intercellular space became narrow.In 4-PBA-L and 4-PBA-H groups,Müller cell numbers were decreased relative to HG group,and cell shape restored long strips,and the intercellular space became wide.MTT assay showed that the survival rate and cell cycle S phase level of Müller cells in HG group were increased relative to control

关 键 词：内质网应激 4-苯基丁酸 MÜLLER细胞 胶质增生 高糖 视网膜病变 

分 类 号：R96[医药卫生—药理学]